The present invention relates to biotechnology with an emphasis on plant biotechnology, and particularly biotechnology affecting the biosynthesis of steroid compounds.
Enhancement of the nutritional or health benefits of oils through genetic engineering is being addressed throughout the agricultural community. Several approaches involve manipulation of already present cellular biosynthetic pathways. Steroid biosynthetic pathways are of current interest, particularly for the enhancement of health benefits from food oils.
Several related U.S. patents address increasing sterol accumulation in higher plants. Those patents include U.S. Pat. No. 5,589,619 xe2x80x9cProcess and Composition for increasing squalene and sterol accumulation in higher plantsxe2x80x9d (accumulation of squalene in transgenic plants by increasing HMGR activity) and U.S. Pat. No. 5,306,862 xe2x80x9cMethod and composition for increasing sterol accumulation in higher plantsxe2x80x9d (increasing HMGR activity to increase plant sterol accumulationxe2x80x94including sterol and cycloartenol, which affects insect resistancexe2x80x94in tobacco, tomato, corn, carrot, soybean, cotton, barley, arabidopsis, guayule and petunia; seeds with elevated sterol/cycloartenol, 7S promoter and CaMV promoters), U.S. Pat. No. 5,365,017 xe2x80x9cMethod and composition for increasing sterol accumulation in higher plantsxe2x80x9d (DNA construct with HMGR-CaMV 35S, transgenic plants, hybrid plants, corn, soy, barley, tomato, Arabidopsis), U.S. Pat. No. 5,349,126 xe2x80x9cProcess and composition for increasing squalene and sterol accumulation in higher plantsxe2x80x9d (increase in squalene and sterol accumulation by increasing HMGR activity in transgenic tobacco, cotton, soybean, tomato, alfalfa, Arabidopsis, corn, barley, carrot and guayule plants), and EP 486290 (enhancement of squalene and specific sterol.[squalene zymosterol, cholest-7,24-dienol, cholest-5,7,24-trienol] accumulation in yeast by increasing HMGR activity in yeast deficient in enzymes that convert squalene to ergosterol).
In those patents, the amount of a protein exhibiting 3-hydroxy-3-methylglutaryl Coenzyme-A reductase (HMGR) activity is typically increased. HMGR widens a xe2x80x9cbottleneckxe2x80x9d near the beginning of a biosynthetic path to steroid production, permitting a higher carbon flux through steroid biosynthetic pathways and resulting in increased sterol accumulation.
U.S. Pat. No. 5,480,805 xe2x80x9cComposition for modulating sterols in yeastxe2x80x9d (enhancement of delta 8-7 isomerase activity-ERG2 enhances accumulation of specific sterols in yeast).
U.S. Pat. No. 5,460,949 xe2x80x9cMethod and composition for increasing the accumulation of squalene and specific sterols in yeastxe2x80x9d (increasing squalene, zymosterol and specific sterols in yeast by increasing HMGR in yeast having decreased erg5 and erg6 activityxe2x80x94Sc and hamster HMGR).
WO 9845457 (SMTI, Erg6 from A.t., corn, yeast; transgenic plants with altered sterol levels_using DNA encoding an enzyme binding a first sterol and producing a second sterolxe2x80x94altered carotenoid, tocopherol, modified FA levelsxe2x80x94HMGR, 5xcex1-reductase, geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, isopentenyl diphosphate isomerase).
Acetate is the metabolic precursor of a vast array of compounds vital for cell and organism viability. Acetyl coenzyme A (CoA) reacts with acetoacetyl CoA to form 3-hydroxy-3 methylglutaryl CoA (HMG-CoA). HMG-CoA is reduced to mevalonate in an irreversible reaction catalyzed by the enzyme HMG-CoA reductase. Mevalonate is phosphorylated and decarboxylated to isopentenyl-pyrophosphate (IPP). Through the sequential steps of isomerization, condensation and dehydrogenation, IPP is converted to geranyl pyrophosphate (GPP). GPP combines with IPP to form farnesyl pyrophosphate (FPP), two molecules of which are reductively condensed to form squalene, a 30-carbon precursor of sterols.
A key enzyme in sterol biosynthesis is 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG-CoA reductase or HMGR). Schaller et al. (Plant Physiol. 109: 761-770, 1995) found that over-expression of rubber HMGR (hmg1) genomic DNA in tobacco leads to the overproduction of sterol end-products (sitosterol, campesterol and stigmasterol) up to 6-fold in leaves. Further, the excess sterol was stored as steryl-esters in lipid bodies. HMGR activity was increased by 4- to 8-fold.
Sterols are derivatives of a fused, reduced ring system, cyclopenta-[a]-phenanthrene, comprising three fused cyclohexane rings (A, B, and C) in a phenanthrene arrangement, and a terminal cyclopentane ring (D) having the formula (I) and carbon atom position numbering shown below: 
where R is an 8 to 10 carbon-atom side chain.
In plants, squalene is converted to squalene epoxide, which is then cyclized to form cycloartenol (4,4,14xcex1-trimethyl-9xcex2,19-cyclo-5xcex1-cholest-24-en-3xcex2-ol). Cycloartenol has two methyl groups at position 4, a methyl group at position 14, a methylene bridge between the carbon atoms at positions 9 and 19 that forms a disubstituted cyclopropyl group at those positions, and includes an 8-carbon sidechain of the formula: CH3CH(CH2)2CHxe2x95x90C(CH3)2. Squalene epoxide can alternatively be converted into pentacyclic sterols, containing five instead of four rings. Exemplary pentacyclic sterols include the phytoalexins and saponins.
Being one of the first sterols in the higher plant biosynthetic pathway, cycloartenol serves as a precursor for the production of numerous other sterols. In normal plants, cycloartenol is converted to predominantly 24-methylene cycloartenol (4,4,14xcex1-dimethyl-9xcex2, 19-cyclo-22,23-dihydro-ergosta-24(28)-en-3-xcex2-ol), cycloeucalenol, (4,14xcex1-trimethyl-9xcex2,19 cyclo-5xcex1-ergosta-24(28)-en-3xcex2-ol), isofucosterol (5xcex1-stigmasta-5-24(28)-dien-3xcex2-ol), sitosterol (5xcex1-stigmasta-5-en-3xcex2-ol), stigmasterol-(stigmasta-5,-22-dien-3xcex2-ol), campesterol (5xcex1-ergosta-5-en-3xcex2-ol), and cholesterol (5xcex1-cholesta-5-en-3xcex2-ol). These transformations are illustrated in FIG. 1.
Although sterols produced by plants, and particularly higher (vascular) plants, can be grouped by the presence or absence of one or more of several functionalities, plant sterols are classified into two general groups herein; i.e., those containing a double bond between the carbon atoms at positions 5 and 6 (delta-5 or xcex945 sterols) and those not containing a double bond between the carbon atoms at positions 5 and 6 (non-delta-5 sterols).
Exemplary naturally-occurring delta-5 plant sterols are isofucosterol, sitosterol, stigmasterol, campesterol, cholesterol, and dihydrobrassicasterol. Exemplary naturally occurring non-delta-5 plant sterols are cycloartenol, 24-methylene cycloartenol, cycloeucalenol, and obtusifoliol. The most abundant sterols of vascular plants are campesterol, sitosterol, and stigmasterol, all of which contain a double bond between the carbon atoms at positions 5 and 6 are classified as delta-5 sterols.
The HMG-CoA reductase enzymes of animals and yeasts are integral membrane glycoproteins of the endoplasmic reticulum. The intact enzyme comprises three regions: a catalytic region containing the active site of the enzyme; a membrane binding region anchoring the enzyme to the endoplasmic reticulum; and a linker region joining the catalytic and membrane binding regions of the enzymes. The membrane binding region occupies the amino-terminal (N-terminal) portion of the intact protein, whereas the catalytic region occupies the carboxy-terminal (C-terminal) portion of the protein, with the linker region constituting the remaining portion. M.E. Basson et al., Mol. Cell Biol., 8(9):3797-3808 (1988).
The activity of HMG-CoA reductase in animals and yeasts is known to be subject to feedback inhibition by sterols. Such feedback inhibition requires the presence of the membrane binding region of the enzyme. See, e.g., G. Gil et al, Cell, 41:249-258 (1985); M. Bard and J. F. Downing, J. Gen. Microbiol., 124:415-420 (1981).
Given that mevalonate is the precursor for sterols and other isoprenoids, it might be expected that increases in the amount or activity of HMG-CoA reductase would lead to increases in the accumulation of both sterols and other isoprenoids.
In mutant strains of the yeast Saccharomyces cerevisiae (S. cerevisiae) having abnormally high levels of HMG-CoA reductase activity, the production of two sterols, 4,14-dimethylzymosterol and 14-methylfucosterol is markedly increased above normal. Downing, et al., Biochem. Biophys. Res. Comm., 94(3): 874-979 (1980).
When HMG-CoA reductase activity was increased by illumination in non-photosynthetic microorganisms, isoprenoid (carotenoid), but not sterol (ergosterol), synthesis was enhanced. Tada, et al., Plant and Cell Physiology, 23(4):615-621 (1982).
WO 9703202 discloses a method for identifying agents modulating sterol biosynthesis using a yeast acetoacetyl CoA thiolase (ERG10) gene linked to a reporter system to evaluate compounds, such as lovastatin and other HMG-CoA synthase inhibitors, that affect cholesterol biosynthesis.
U.S. Pat. No. 5,668,001 teaches a recombinant avian HMG-CoA synthase preparation useful for evaluating drugs that inhibit cholesterol biosythesis.
JP 09121863 discloses a plant with increased 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) activity as a result of increasing the expression of a mutant protein kinase gene that regulates expression of the HMGR gene. The increased HMGR activity increases squalene, zymosterol, cholesta-7,24-dienol and cholest-5,7,24-trenol accumulation in yeast with ERG5 and ERG6 mutants.
EP 480730 xe2x80x9cPlant-sterol accumulation and pest resistance-by increasing copy number of 3-hydroxy-3-methyl glutaryl coenzyme-A reductase gene in tobacco, tomato and corn.
WO 9913086 xe2x80x9cHuman Delta 7-sterol reductase polypeptide-useful for diagnosis or treatment of genetic defects e.g. hereditary Smith-Lemli-Opitz syndromexe2x80x9d teaches making and using the recombinant polypeptide with humans.
Chappell et al. U.S. Pat. No. 5,589,619 teaches that transformation of higher plants with truncated HMG-CoA reductase enhanced the production of squalene, cycloartenol and certain sterols, particularly compounds having unsaturations at the 5-position. Several intermediate sterols as are shown in FIG. 1 were also produced. It would be beneficial if the production of sitosterol and stigmasterol could be enhanced while lessening the accumulation of the intermediate sterols. The present invention provides avenues for enhancing production of sitosterol and stigmasterol and lessening the accumulation of the intermediate sterols.
Gonzalez et al. (Abstract of poster at Third Terpnet Meeting of the European Network on Plant Isoprenoids, May 29-30, 1997, Poitiers, France) over-expressed the Arabidopsis HMGR cDNA (hmg1 and hmg2) and found sterol overproduction with hmg1 only. They used two forms of the hmg1 gene, a full-length form and a truncated form containing only the catalytic domain. HMGRs have three domains, an N-terminal membrane spanning domain, a short linker domain, and a C-terminal catalytic domain. In this case the transgenic plants were also Arabidopsis. The difference between the full-length and truncated forms was a greater accumulation of pathway intermediates in the case of the truncated form. More importantly, the intermediates demonstrated as accumulating were cycloartenol, 24-methylenecycloartanol and obtusifoliol.
Finally, U.S. Pat. Nos. 5,365,017 and 5,306,862, both assigned to Amoco Corp., disclose a method for increasing sterol accumulation in plants by increasing the copy number of a gene having HMG-CoA reductase activity. These inventions disclose a method using hamster truncated HMGR that consisted of the catalytic domain and the linker domain. According to the claims the linker domain was essential for activity. They also demonstrated a greater accumulation of pathway intermediates such as cycloartenol.
The present invention relates to transgenic plants and their progeny having improved nutritional characteristics. More particularly, the present invention relates to transgenic plants and their progeny, the storage organs (e.g. seed, fruit and vegetable parts) of which contain modified levels of steroid compounds, such as (i) elevated levels of beneficial phytosterols (e.g., sitosterol), phytostanols (e.g., sitostanol), and esters thereof, relative to an otherwise identical plant transformed only with a truncated HMG-CoA reductase gene or a wild-type plant, and (ii) reduced levels of steroid pathway intermediate compounds (e.g. one or more of squalene, cycloartenol, 24-methylene cycloartenol, obtusifoliol, stigmasta-7-enol and campesterol) in their storage organs relative to an otherwise identical transgenic plant transformed only with a truncated HMG-CoA reductase gene. Nucleic acid sequences encoding enzymes that affect the biosynthesis and accumulation of steroid compounds in plants (HMG-CoA reductase and a steroid pathway enzyme), and methods for using these sequences to produce such transgenic plants, are also provided. These methods comprise, for example, introducing into cells nucleic acid sequences encoding enzymes that affect the levels of accumulated steroid pathway end products.
The present invention contemplates a recombinant construct or a recombinant vector that contains 2 DNA sequences. The first encodes a polypeptide exhibiting 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase activity. The second DNA sequence encodes a polypeptide exhibiting the activity of another steroid pathway enzyme. Each polypeptide-encoding DNA sequence is operably linked in the 5xe2x80x2 to 3xe2x80x2 direction to a promoter and a transcription termination signal sequence independent of the other sequence. The promoter is located upstream and the termination sequence downstream of each polypeptide-encoding DNA sequence. The second DNA sequence encoding a steroid pathway enzyme can code for a squalene epoxidase enzyme, a sterol methyl transferase I enzyme, a sterol C4-demethylase enzyme, a obtusifoliol C14xcex1-demethylase enzyme, a sterol C5-desaturase enzyme, or a sterol methyl transferase II enzyme. It is contemplated that HMG-CoA reductase and the steroid pathway enzyme activity comes from a mutant or truncated form of those enzymes, such as a truncated HMG-CoA reductase lacking the transmembrane region while retaining a functional catalytic domain. Examples of such preferred HMG CoA reductases include the truncated rubber and Arabidopsis HMG CoA reductases disclosed herein.
Preferably, the regulatory function of a promoter is substantially unaffected by cellular levels of squalene such as the CaMV 35S promoter. In one aspect, a promoter is seed-specific. In another aspect, a promoter is derived from a species in a different order from a host cell. In another aspect, the HMG-CoA reductase or steroid pathway enzymes is from a species in a different order from the order that of the host cell. The invention contemplates a construct or recombinant vector having more than one DNA sequence encoding a steroid pathway enzyme that do not have to be under the control of the same promoter. Preferably, a recombinant vector is a plant expression vector.
In another aspect of the invention, a transformed host cell comprises a recombinant construct or vector as described above. Preferably, a host cell is plant cell, preferably that plant cell is from canola, soybean, corn, maize, tobacco, cotton, rape, tomato or alfalfa. The invention contemplates a host cell in a cell culture, plants derived from transformed host cells, and storage organs (seeds, fruits and vegetable parts) from transgenic plants.
In addition to contemplating transgenic plants and seeds, the invention contemplates transgenic plant seeds capable of germinating into a transgenic plant and mutants, recombinants, genetically engineered derivatives thereof and hybrids derived therefrom. The plant over-accumulates steroid pathway products relative to a native, non-transgenic plant of the same strain, wherein said mutants, recombinants, genetically engineered derivatives thereof and hybrids derived therefrom maintain the ability to overaccumulate steroid pathway products.
The invention contemplates a process of increasing the formation of steroid pathway products in a transformed host cell as compared to an otherwise identical non-transformed host cell. Contemplated processes use the described recombinant constructs and vectors to transform host cells, then growing the host cells or regenerating transgenic plants therefrom.
In one aspect of the invention, the genome of a contemplated plant, its progeny, seeds or cell culture, comprises introduced DNA encoding an HMG-CoA reductase activity and introduced DNA encoding a steroid pathway enzyme that is a squalene epoxidase enzyme, a sterol methyl transferase I enzyme, a sterol C4-demethylase enzyme, a obtusifoliol C14xcex1-demethylase enzyme, a sterol C5-desaturase enzyme, or a sterol methyl transferase II enzyme. The storage organs of such a plant contain an elevated level of total accumulated sterol, compared to storage organs of an otherwise identical plant, the genome of which does not comprise said introduced DNA. Further, the storage organs of the plant contain a reduced level of squalene, cycloartenol, 24-methylene cycloartenol, obtusifoliol, stigmasta-7-enol, or campesterol compared to the seeds of an otherwise identical plant or a plant comprising an introduced DNA encoding an HMG-CoA reductase enzyme.
The invention contemplates a method of producing a plant that accumulates an elevated level of sterol pathway products compared to a corresponding plant comprising no introduced DNA encoding a peptide, polypeptide, or protein that affects the biosynthesis and accumulation of a sterol pathway product, comprising sexually crossing plants to arrive at a plant comprising nucleic acid encoding an HMG CoA reductase and a steroid pathway enzyme, including crosses with a nurse cultivar. The plants, including apomicitic plants, uniform populations of the plants and their seeds and parts other than seeds are contemplated.
Another aspect of the invention is oils containing at least one sterol pathway product, extracted from the seeds of a contemplated plant. Preferably sitosterol, at least one sitosterol ester, or mixtures thereof, comprise at least about 57% by weight of the total sterol compounds of a contemplated oil. Preferably sitosterol, that at least one sitosterol ester, or mixtures thereof, comprise at least about 0.08% of the dry weight of a contemplated seed. Preferably, the oil has a reduced amount of squalene, cycloartenol, 24-methylene cycloartenol, obtusifoliol, stigmasta-7-enol, campesterol, or combinations thereof, compared to oil from a corresponding transgenic plant that does not contain introduced DNA encoding a squalene epoxidase enzyme, a sterol methyl transferase I enzyme, a sterol C4-demethylase enzyme, a obtusifoliol C14xcex1-demethylase enzyme, a sterol C5-desaturase enzyme, a sterol methyl transferase II enzyme, or mixture thereof; wherein the reduction is in the range of from about 10% to about 100%.
Sitosterol ester compositions derived from transgenic plants of the present invention, their progeny or their seeds are also contemplated, preferably wherein an esterifying fatty acid has 2 to 22 carbon atoms in the main chain.
A further aspect of the invention is cholesterol-lowering compositions comprising contemplated oils and sitosterol ester compositions. Another further aspect of the invention is foods, food ingredients, or food compositions comprising contemplated oils.
Still further, the invention contemplates pharmaceutical compositions comprising a cholesterol-lowering effective amount of a contemplated oil, and a pharmaceutically acceptable carrier, excipient, or diluent.
A method of lowering the plasma concentration of low density lipoprotein cholesterol is contemplated, comprising orally administering to a human or animal subject an effective amount of an above composition. Also contemplated is a method of treating or preventing an elevated plasma concentration of low-density lipoprotein cholesterol, comprising orally administering to a human or animal subject an effective amount of a contemplated composition.
A related aspect of the invention is a method of making a food additive composition, comprising obtaining oil containing a sterol pathway product compound from seed of a contemplated transgenic plant and mixing the oil with an edible solubilizing agent, an effective amount of a dispersant, and optionally, an effective amount of an antioxidant.
Novel forms of two sterol pathway enzymes and the nucleic acids that encode them are disclosed: an Arabidopsis enzyme having nucleic acid similarity to a squalene epoxidase, and an Arabidopsis enzyme having nucleic acid similarity to an obtusifoliol C14xcex1-demethylase enzyme. Thus, the invention contemplates an isolated DNA molecule having a nucleotide sequence of disclosure SEQ ID NO: 4, 6, 8, 10, 14, 15, 17 or the complements thereof. Also contemplated is a nucleotide sequence that hybridizes to the nucleotide sequence of SEQ ID NO:4, 6, 8, 10, 14, 15, 17 or their complements under a wash stringency equivalent to 0.5X SSC to 2X SSC, 0.1% SDS, at 55-65xc2x0 C., and that encode a polypeptide having squalene epoxidase or obtusifoliol C14xcex1-demthylase enzymatic activity. Preferably, that enzymatic activity is substantially similar to that of a disclosed squalene epoxidase or obtusifoliol C14xcex1-demethylase, respectively. By substantially smiliar is meant having enzymatic activity differing from that of the disclosed enzymes by about 30% or less, preferably by about 20% or less, and more preferably by about 10% or less when assayed by standard enzymatic assays. Also contemplated is a nucleotide sequence encoding the same genetic information as said nucleotide sequence of SEQ ID NO: 4, 6, 8, 10, 14, 15, 17 or their complements or that hybridize as described above, but which is degenerate in accordance with the degeneracy of the genetic code. Recombinant constructs, vectors and transformed host cells comprising the novel isolated and purified nucleic acid sequences are also contemplated. In one embodiment, the vector is a plant vector and the host cell is a plant cell. Methods of producing the disclosed squalene epoxidase or obtusifoliol C14xcex1-demethylase enzymes are also contemplated comprising culturing a transformed host cell for a time and under conditions conductive to the production of the squalene epoxidase or obtusifoliol C14xcex1-demethylase enzyme, and recovering the produced squalene epoxidase or obtusifoliol C14xcex1-demethylase enzyme.
Yet another aspect provides any of the above described transformed host cells, further comprising a recombinant construct or expression vector encoding a tocopheral synthesis pathway enzyme, and in particular, S-adenosylmethionine-dependent xcex1-tocopherol methyltransferase. Also included are plants, seeds and storage organs comprising the transformed host cells.
Another aspect provides, a process of increasing the formation of steroid pathway products and tocopherols in a transformed host cell as compared to an otherwise identical non-transformed host cell comprising (1) transforming a host cell with a recombinant vector comprising (a) as operably linked components in the 5xe2x80x2 to 3xe2x80x2 direction, a promoter, a DNA sequence encoding a first polypeptide having 3-hydroxy-3-methylglutaryl-Coenzyme A reductase enzyme activity, and a transcription termination signal sequence; and (b) as operably linked components in the 5xe2x80x2 to 3xe2x80x2 direction, a promoter, a DNA sequence encoding at least one polypeptide having steroid pathway enzyme activity selected from the group consisting of squalene epoxidase enzyme activity, sterol methyl transferase I enzyme activity, sterol C4-demethylase enzyme activity, obtusifoliol C14xcex1-demethylase enzyme activity, sterol C5-desaturase enzyme activity, and sterol methyl transferase II enzyme activity, and a transcription termination signal sequence; (2) transforming the host cell of (1) with a recombinant vector comprising as operably linked components, a promoter, a DNA sequence encoding a tocopherol synthesis pathway enzyme, and a transcription termination sequence; and (3) regenerating said transformed plant cell into said transgenic plant.